The present invention relates to a liquid chromatograph/mass spectrometer(LC/MS) that separates and analyzes liquid mixture, particularly relates to a protein analysis (proteomics) system and a glycan analysis (glycomics) system that on-line analyze organism-related substance mixed liquid to which pretreatment such as enzyme digestion is applied. In a field of the organism-related substance analysis, it is regarded as important to separate and analyze a sample of an extremely minute amount.
LC/MS has been used for analyzing an organismal sample of a minute amount. However, as an amount of a sample is reduced, high separation (high concentration) in LC (the liquid chromatograph) and the enhancement of the efficiency of ionization in an LC/MS interface have been recognized as important. Spray ionization such as electrospray is applied to the ionization technique of the interface and it is known that the less a liquid flow rate is, the more the efficiency of ionization tends to be enhanced. Electrospray ionization is described in Analytical Chemistry, Vol. 65, 1993, pp. 972A to 986A for example. In the interface using electrospray ionization, high voltage of approximately a few kV is applied between liquid taken in a spray capillary and an ion intake port (a metallic sampling orifice) of a mass spectrometer. Charged fine droplets leap out of liquid at the end of the spray capillary toward the ion intake port of the mass spectrometer and gaseous ions are generated based upon the charged fine droplets by the effect of evaporation.
In the meantime, the reduction of a liquid flow rate in LC and high separation can be compatible. Therefore, a new interface based upon nanoLC in which LC separation occurs at a little flow rate at which a liquid flow rate is at a level of a nanoliter and spray ionization using a nanospray having small-sized configuration in which efficient ion generation is enabled at a little flow rate has been used. The nanospray is described in International Journal of Mass Spectrometry and Ion Processes, Vol. 136, 1994, pp. 167 to 180 for example. In an interface using the nanospray, a spray capillary the end of which is tapered is used. The nanospray is acquired by making a spray capillary of an electrospray fine and it is considered that an ion generation principle is the same. However, when the surface area of liquid exposed to the end of the spray capillary is large, a liquid flow rate and an amount of evaporation at the end of the spray capillary becomes equal and ionization becomes unstable or is stopped. This problem becomes remarkable as a liquid flow rate decreases and the size of the end of the spray capillary is required to be miniaturized corresponding to the reduction of a flow rate. Therefore, the size at the end of the spray capillary of the nanospray is often required to be in units of a micron and the spray capillary is often made of fused silica and glass. For a metallic spray capillary, as there is a problem in working, the size of the end is currently larger than 30 μm. Therefore, at an extremely little flow rate of 200 nanoliter/minute or less, a spray capillary made of fused silica and glass is often used.
In U.S. Pat. No. 5,572,023, description related to a column-integrated LC/MS interface in which an LC separation column is plugged into a spray capillary the end of which is tapered is found. In this configuration, the end of the separation column and the end of the spray capillary are substantially coincident. Therefore, an effluent separated in the LC column is sprayed without being substantially influenced by diffusion. As a result, a highly separated (concentrated) sample is efficiently ionized and can be analyzed in a mass spectrometer. Besides, in Nature Biotechnology, Vol. 17, 1999, pp. 676 to 682, an example that a reverse-phase column is first plugged into a spray capillary made of fused silica and next, an ion exchange column is plugged is described. In this example, two-dimensional LC separation can be realized by a simple system by changing the composition of mobile-phase liquid. For ionization, electrospray ionization which is one type of spray ionization is often used.
In the electrospray ionization, high voltage is required to be applied between liquid at the end of the spray capillary and an ion intake port of a mass spectrometer. Therefore, the spray capillary is fixed to a metallic union (see FIG. 2) and an electrode is often installed on the metallic union. As acid such as formic acid is added to the mobile-phase liquid, the metallic union and the liquid at the end of the spray capillary electrically conduct. The mobile-phase liquid is taken in the metallic union at a fixed flow rate from a capillary on the upstream side by a pump system and is taken in the spray capillary.
The spray capillary integrated with the separation column described above is often required to be replaced. 1) The reason is that nonvolatile materials deposit by the evaporation of the liquid at the end and may clog the spray capillary. 2) The reason is that the separation column is clogged with the materials of the sample. 3) The reason is that the separation column is contaminated. The spray capillary itself is often made of fused silica and glass, the end of the spray capillary has size in units of a micron and is very easily damaged. Actually, the end of the capillary is damaged only by being touched to a someone in replacing spray capillaries. The damaged capillary cannot be used. Therefore, close attention is required in replacing spray capillaries.
Then, as shown in FIG. 2, a trial that a spray capillary 2 and a separation column 1 are separately formed and are connected via a metallic union 3 is made. For example, in Analytical Chemistry Vol. 74, 2002, pp. 4725 to 4733, an example using this configuration is given. In this configuration, high voltage is applied between the metallic union 3 and an ion intake port (not shown) of a mass spectrometer so as to generate ions.